The arthropod-vectored alphavirus eastern equine encephalitis virus (EEEV) is one of the most virulent viruses endemic to the United States (US), causing devastating disease and mortality in a high percentage of infected humans and equines. Due to its extreme neurovirulence, widespread distribution in the eastern US and potential for use as a bioweapon, it is categorized as a Select Agent virus and NIH Category B Priority Pathogen. Yet, no antiviral drugs or licensed human vaccines are available to combat this virus and it is critically understudied. The virus-receptor interaction represents a target fo antiviral therapeutics and can be used in rational design of vaccine vectors. Heparan sulfate (HS) is a sulfated polysaccharide identified as an attachment receptor for multiple alphavirus and flavivirus laboratory strains but its relevance to viruses in nature is in question because its use as a receptor is possibly an artifact of adaptation to in vitro growth. To address this question, we compared the E2 attachment protein amino acid sequence between a HS binding EEEV laboratory strain and over 60 EEEV strains, including viruses sequenced directly from unamplified field samples of animal tissues. This analysis confirmed that HS is a receptor for naturally circulating EEEV. By mutagenesis of the EEEV attachment protein eliminating the HS binding phenotype, we determined that HS binding was responsible for multiple unique aspects of EEEV disease in vertebrates including extreme neurovirulence, limited spleen replication and suppression of cytokines/chemokines involved in innate and adaptive immune responses. This suggests that HS binding promotes EEEV replicative fitness in vivo. The experiments in this application will investigate the role of HS binding in EEEV disease and tissue targeting in vertebrate and mosquito hosts identifying points in the arbovirus replication/transmission cycle vulnerable to therapeutic intervention and provide the basis for the rational design of anti-EEEV vaccines.